Population shifts in some faeces and rumen bacteria profiles and subsequent blood LPS and lactate concentrations in lambs in the early period of subacute ruminal acidosis

Abstract Background It is known that ruminal acidosis can induce harmal population shifts in some ruminal bacteria profiles. However, there is little information related to alterations in faecal and ruminal bacterial communities and relevant serum lipopolysaccharide (LPS) in sheep with subacute ruminal acidosis (SARA). Objectives This study aimed to investigate alterations in the defined faecal and ruminal bacteria profiles and serum LPS and blood lactate concentrations in lambs with empirically induced SARA. Methods Fifteen lambs were served and undergone to induce SARA during a 7‐day period. Faecal and ruminal samples were taken to measure the pH and to perform the bacteriological works at 0 (just before induction), 8, 9, and 10 days of the challenge. Blood samples were collected to determine the serum LPS and lactate levels. The rumen and faecal samples were cultured to specify colony‐forming units (CFU) for Escherichia coli, Streptococcus Group D (SGD), and lactic acid bacteria (LAB). Results Serum LPS value had no significant increase in the affected lambs with SARA. Significant increasing trends were observed in faecal E. coli and LAB populations (p < 0.01). Rumen bacteriology revealed a rising trend for LAB and a falling trend for SGD populations (p < 0.01). Conclusion : Unlike cattle, LPS appears to be of minor importance in the pathogenesis of SARA in sheep. The increased ruminal and faecal LAB (4.00 × 107 CFU/ml or g) are proposed as valuable biomarkers for improving nutritional strategy and screening SARA in lambs.


INTRODUCTION
Ruminal acidosis is a prevalent nutritional disorder in ruminants. This disorder increases herd mortality, dramatically reduces weight gain, and complicates nutritional strategies in cattle and sheep. Subacute ruminal acidosis (SARA), also known as subclinical acidosis, is economically more important than its acute form, and it is often able to affect a significant proportion of herd (Constable et al., 2017). SARA has been defined as a sequel of feeding ruminants (formerly on predominantly forage-based rations) with high-grain rations (Smith, 2015).
Several studies have focused on the epidemiology and pathophysiology of the disease in cattle, sheep, and goat (Bramley et al., 2008;Lee et al., 2019;F. Li et al., 2017;Plaizier et al., 2017a). SARA is generally defined as a depressed rumen pH value for extended periods (Kleen & Cannizzo, 2012;Plaizier et al., 2008). It has been considered by several researchers as a rumen with pH below 5.80 (5.80-5.20) (Morgante et al., 2009;Sharma et al., 2009). Rumen pH has been reported to vary in SARA, with the lowest value recorded 2-4 h following concentrate consumption (Harrison et al., 1989). As a part of SARA pathogenesis, the escape of large amounts of fermentable carbohydrates from the rumen and small intestine could lead to extensive fermentation in the hindgut during SARA. Hindgut fermentation was also shown to result in reduced digesta and faecal pH in SARA (Gressly et al., 2011;. Furthermore, S. Li et al. (2012) claimed that hindgut fermentation might alter the faecal bacterial communities during SARA.
Several experiments have been conducted on rumen Plaizier et al., 2017a) and faecal (Mao et al., 2012) bacteria communities in dairy cows during grain-induced SARA; one of these studies reported a dominant rumen flora of Streptococcus bovis and Escherichia coli . Despite the rumen and faecal bacteriological studies during SARA in cow, little information is available on changes in rumen and faecal bacterial density and diversity in sheep or lambs with SARA (F. Li et al., 2017).
The E. coli is a predominant rumen bacterial flora at the normal pH value. Following consumption of fermentable carbohydrates and reduction of the rumen pH, due to overgrowing in Streptococcus spp.
proliferation and excessive volatile fatty acids (VFAs) accumulation in the rumen, it is expected to kill the Gram-negative rumen flora such as E. coli and subsequently blood lipopolysaccharide (LPS) increment (Constable et al., 2017;). On the other hand, it has been shown that the numerous species of lactateproducing and lactate-using bacteria can adapt to a decreased rumen pH (Smith, 2015).
Although the ruminal pH assessment is a valuable method for diagnosing SARA, it has been shown that the changes in the communities and the abundances of rumen bacteria are more stable than the ruminal pH changes over 2-4 h after concentrate feeding in lambs (F. Li et al., 2017). It has also been reported that abundances of several rumen cellulolytic bacteria could be used as a potential biomarker for SARA screening in lamb flock (F. Li et al., 2017). Therefore, a better understanding of the rumen and faecal bacterial structures during SARA in sheep can be conducive to diagnosing or specifying the risk of SARA and to developing a better nutritional strategy.
The major pathogenesis of SARA is related to VFAs accumulation, pH depression, bacterial population changes, and subsequently LPS releasing into the bloodstream (Constable et al., 2017;Plaizier et al., 2008). It has been revealed that SARA induction via feeding high-grain rations is associated with elevated levels of free bacterial LPS in the ruminal and intestinal digesta in dairy cows . The LPS or endotoxin could translocate into blood and result in many complications, including inflammatory responses, ruminal stasis, poor blood supply in tissue, and weakness (Pulina & Bencini, 2004). However, blood LPS changes and its association with the rumen and faecal bacterial perturbations during SARA in sheep are still unclear. Therefore, the current study aimed to (1) investigate the change in the density of certain ruminal and faecal bacterial communities and (2) evaluate their relationship with the blood LPS and lactate concentrations in experimentally induced SARA in lambs.

Study periods
The study course comprised a 1-week on basal diet and 1 week of induction (adaptation) period which were followed by a 3-day period of SARA status.

Period on basal diet
After determining the dry matter (DM) content of dietary ingredients (crushed corn 89.40%, alfalfa 86.20%, and wheat straw 92.20%), in the first week of the study, the lambs were individually fed with a basal diet at roughly 3.00% body weight with a 1:4 ratio of corn to forage (220 g crushed corn, 430 g straw, and 400 g alfalfa) on a DM basis. Water access was free. The diurnal rations were offered in three servings at 8:00, 14:00, and 19:00 h, such that the corn was fed in the morning (8:00) and in the afternoon (14:00) (Nikvand et al., 2020).

Induction (adaptation) period and sampling
Just before the induction (zero day), as the pre-treatment sampling, the blood, rumen fluid, and faecal samples were taken from the lambs.
Afterwards, the crushed corn was gradually added to the basal diet on a daily basis at 5.00% weight of DM ration (42.00 ± 8.00 g), and the same amount of straw was simultaneously deducted. Each lamb was individually fed with crushed corn (Nikvand et al., 2020). In all animals, on the 7th day of SARA induction, when the amount of crushed corn increased to 52.80% (566 ± 92.00 g) of the ration, ruminal pH was reduced to a range of 5.60-5.70 between 4 and 7 h after morning feeding (for at least 3 h per day); this pH range represented SARA (Constable et al., 2017;Morgante et al., 2009;Penner et al., 2009). A rumen pH threshold below 5.80 has been widely applied to determine SARA severity in sheep .
Rumen sampling: After rumen content sampling with an orogastric tube (Lee et al., 2019;F. Li et al., 2017), pH was immediately specified by a pen type digital pH meter (AZ8686; AZ Instrument Corp., Taichung, Taiwan). The ruminal pH was measured twice a day between 4 and 7 h after morning feeding during induction and SARA periods. To avoid oral bacteria and saliva contamination of the rumen samples, a disinfected large-bore orogastric tube (1.50 cm of the inside diameter and 40.00 cm in length) was primarily inserted in the upper oesophagus. Next, a disinfected small-bore stomach tube (1.00 cm of the inside diameter) was passed through the larger tube into the rumen. The first amounts (approximately 50.00 ml) of the gushed out fluid samples were further discarded due to the possible oral bacteria and saliva contamination. It is notable that prior to use, the orogastric tubes were washed with Betadine iodine 10.00% and then rinsed with sterile saline solution.
Faecal sampling: A faecal specimen was directly obtained from the rectum using disposable gloves. In this way, 10 g of the specimen was transferred into a sterile falcon tube and referred to the laboratory for bacteriological testing; also, 5 g was mixed with the same amount of distilled water, and its pH was immediately determined by the digital pH meter (Johnson & Rossow, 2018).
Blood sampling: Two blood samples were also taken from the jugular vein. To measure the blood pH and certain biochemical variables, a heparinized blood sample was taken by a 2 ml-sterile syringe manually soaked with 0.05 ml of sodium heparin (125 IU/ml blood; Caspian Co., Rasht, Iran). Venous blood gas analysis has been applied to evaluate some blood variables regarding ruminal acidosis in cows (Marchesini et al., 2013). For serum separation, after clotting, anticoagulant-free blood samples were centrifuged at 3000 rpm for 10 min; then, the harvested sera were stored in 1.50 ml sterile micro-tubes and frozen at -70˚C until further use.

SARA period
Over the 3 consecutive days of SARA (which were days 8, 9, and 10 of the challenge), each lamb was fed with 566 ± 92 g of crushed corn, 98 ± 16 g of wheat straw, and 400 g of alfalfa. To assess the rumen bacteriology, 10 ml of the rumen fluid sample was daily taken from each animal, transferred to a sterile falcon tube, and referred to bacteriological evaluations in less than 1 h. The pH was determined with the rest of the ruminal sample. The blood samples were further obtained to analyze the blood biochemical parameters. Every 3 days of the SARA period, a faecal specimen was taken from each lamb for pH determination and bacteriology.

Bacteriology of rumen samples
The

Bacteriology of faecal samples
At first, 1 g of a faecal sample was mixed with 9 ml of sterile PBS solution to obtain a homogeneous and diluted solution at 1:10 (10 −1 ) level.
Then, 50 µl of this dilution was inoculated and cultured in each of EMB, BEA, and MRS media. The faecal samples (50 µl), diluted at 1:100,000 (10 −5 ) level, were further cultured in TSA medium for total anaerobic bacterial count. The culture conditions and CFU/g calculation for faecal samples in different media for rumen fluid samples were the same as mentioned above (Markey et al., 2013).

Blood biochemical analyses
Blood biochemical variables such as lactate, bicarbonate, anion gap, and blood pH were immediately measured after sampling by use of a blood gas analyzer and its cartridge pack (EDAN; Edan Instruments Inc., Shenzhen, China) (Johnson & Rossow, 2018

Data analysis
Shapiro-Wilk test showed that some but not all data had a normal distribution. For the normally distributed data, a repeated measure analysis of variance (ANOVA) was employed to evaluate the overall trend of data changes over the four time points of sampling; these time points consisted of days 0 (just before induction), 8, 9, and 10 of challenge in the studied lambs. For abnormally distributed data (e.g., SGD bacterial data), a non-parametric Friedman test was also employed.
Using Pearson test, the correlations of the serum LPS and blood lactate changes with the rumen and faecal bacterial population were also calculated. The level of significance was set to p < 0.05. The analyses were performed using SPSS statistic software (version 24.0; IBM Corp., Armonk, USA).

Ruminal and faecal pH variations
According to the results, on the seventh day of induction of SARA, the rumen pH of all lambs decreased to a range of 5.60-5.70, which was considered SARA. Statistical analysis showed that the changes in the faecal pH of the lambs were associated with a significant falling trend during days 0 (just before induction), 8, 9, and 10 of the challenge (p < 0.01), (Table 1); therefore, on the second and third days of SARA, the mean faecal pH was reduced to 6.29 ± 0.20. A strong positive correlation was observed between the mean rumen pH changes and the mean faecal pH changes during the times of induction and SARA phases in the studied lambs (r = 0.974, p < 0.001).

Blood parameter variations
Repeated measure ANOVA test, showed that induction of SARA did not significantly change the serum LPS levels at either time points (days 0, 8, 9, and 10 of the challenge) in the studied lambs (Table 2). Based on repeated measure ANOVA, no significant changes were detected in the studied lambs regarding blood lactate, bicarbonate, anion gap, and pH values at either time points during the early period of SARA (Table 2).

Rumen bacteriology results
The data analysis showed a significant falling trend in ruminal anaerobic bacterial count over the early period of SARA (p < 0.01).

F I G U R E 1
Changes in the rumen bacterial counts (CFU/ml) × 10 4 (means) in the studied lambs with induced-SARA. LAB, Lactic acid bacteria; SGD, Streptococcus group D; T0, before induction; Ts8-Ts10, SARA period Different superscript letters on each time point denote significant differences among times (p < 0.01).
Rumen bacteriology revealed a rising trend for LAB and a falling one for SGD population (p < 0.01), (Table 3). Rumen E. coli population slightly increased during this short period of SARA (p > 0.05) ( Figure 1).

Faecal bacteriology results
Significant falling trends were found in faecal anaerobic bacterial densities during early period of SARA (p < 0.01). Non-parametric Friedman test showed that the faecal SGD populations non-significantly increased on days 8, 9, and 10 of SARA in comparison with day 0 (p > 0.05) (Figure 2). A significant rising trend was further seen in LAB counts during SARA (p < 0.01). The faecal E. coli count was associated with a significant increase on days 9 and 10 of SARA compared to day 0 (p < 0.01) ( Table 4).
Pearson correlation test showed no significant association between serum LPS levels with the rumen and faecal E. coli counts and other rumen bacterial populations. Additionally, no significant correlation was observed between blood lactate level and rumen bacterial changes over the course of the experiment.

TA B L E 2
Mean ± SD serum lipopolysaccharide (LPS) and blood lactate, bicarbonate, anion gap, and pH values of studied lambs during subacute ruminal acidosis (SARA)

Blood parameters
Before induction Days of SARA Note: Different superscript letters in each row denote significant differences among times (p < 0.01). Abbreviations: AnTBC, anaerobic total bacterial count; LAB, lactic acid bacteria; SGD, Streptococcus group D.

DISCUSSION
There is no research regarding the changes in rumen bacterial community in the context of rumen pH changes and its association with blood LPS in sheep on a SARA induction diet. It has been shown that SARA in cattle is correlated with change in the ruminal and faecal bacterial community and production of LPS (Plaizier et al., 2017a). It has further been reported that rumen-derived bacterial LPS plays a prominent role in the pathogenesis of SARA in cattle (Guo et al., 2017;Plaizier et al., 2012).
Following the induction period, a reduction occurred in the rumen pH to the range of 5.60-5.70, which is indicative of SARA in the studied lambs. In this regard, a rumen pH threshold value of 5.8 was con-sidered to define SARA in sheep , goats (Dong et al., 2013), and dairy cows (Mao et al., 2012). The results showed that the decrease in the rumen pH of lambs to 5.70-5.60 reduced the faecal pH from 7.30 ± 0.20 to 6.29 ± 0.20 on the second and third days of SARA. Supporting our results, Mao et al. (2012) reported a reduction in faecal pH from 7.15 to 6.40 during SARA in dairy cows.
This may be attributed to hindgut fermentation of an escaped massive unstructured carbohydrate from the rumen (Gressley et al., 2011) Induction of a short period of SARA did not significantly change the serum LPS value over the trial in the studied lambs. In agreement with our study, a previous study reported that feeding with two diets containing 50% and 25% concentrate did not significantly change the serum LPS values in goats (Huo et al., 2014). It has been also F I G U R E 2 Changes in the faecal bacterial counts (colony-forming units [CFU]/g) × 10 4 (means) in the studied lambs with induced-SARA. LAB, Lactic acid bacteria; SGD, Streptococcus group D; T0, before induction; Ts8-Ts10, SARA period Different superscript letters on each time point denote significant differences among times (p < 0.01).
claimed that there is limited evidence of increased circulatory LPS during acidosis, which might be ascribed to measurement errors or rapid blood clearance . In conflict with our results, an increased plasma LPS value was further reported following graininduced SARA in Holstein dairy cows (Guo et al., 2017;Khafipour, Li, et al., 2009). The rumen-derived endotoxins are mainly metabolized by hepatic macrophages (Andersen, 2003;Emmanuel et al., 2007). In dairy cows, the bovine liver function decreased following hepatic lipidosis, which is quite frequent (Andersen, 2003); in the authors' opinion, this may be a possible reason for the difference between sheep and cows regarding the ability of the liver to remove blood LPS. Interestingly, however, SARA did not increase serum LPS values in the studied lambs; it may also be attributed to the short duration of SARA in the studied lambs. Therefore, this necessitates more research for a more definitive conclusion.
The SARA induction did not lead to significant changes in lactate, bicarbonate, anion gap, and blood pH values in the studied lambs.
Similar to our study, previous investigations reported no significant changes in blood lactate and pH values in dairy cows with SARA (Daschner et al., 2015;. It was previously observed that blood lactate, bicarbonate, and pH levels were within normal ranges during SARA in cattle (Brown et al., 2000), which is in agreement butyric, and a small number of lactic acids, are metabolized to some beneficial energy metabolites in the rumen wall and liver after being absorbed; accordingly, they cannot affect the blood acid-base-related components, including pH, bicarbonate, and anion gap (Constable et al., 2017).
Our study showed that the density of ruminal anaerobic bacteria significantly decreased within SARA in lambs, which is consistent with the results of Plaizier et al. (2017b). They observed reductions in the richness and diversity of ruminal bacteria in SARA challenge in dairy cows. Rumen bacteriology revealed an increase in LAB, a decrease in SGD, and no significant changes in E. coli populations in the studied lambs. Some of the foregoing findings were supported by Goad et al. (1998), who reported elevated ruminal Lactobacilli counts in steers on a SARA induction diet. A highly abundant Lactobacillus population was further observed in cows fed on SARA-inducing diets (Wang et al., 2014). In accordance with the present results, a reduction in rumen Streptococcal population was reported in dairy cows on a SARAinducing diet (Mao et al., 2013). Despite the significant increase in the rumen LAB population, either producers or utilizers of lactate, no notable changes were found in the blood lactate level of the studied lambs. This is possibly explained by the fact that major lactate-utilizing bacteria such as Megasphaera elsdenii and Selenomonas Ruminantium are still not suppressed in the rumen pH of 5.70-5.60. (Constable et al., 2017;McCann et al., 2016;Meissner et al., 2010).
The abundance of ruminal fluid E. coli isolated from the current studied lambs on a SARA-inducing diet did not significantly change from before the induction (1.60 × 10 7 ) to the third day of SARA (2 × 10 7 CFU/ml). It may be related to the short duration (3 days) of SARA. Similar findings were reported by Khafipour et al. (2011) in dairy cows with SARA. Serum LPS levels might result from the lysis of rumen Gramnegative bacteria other than E. coli (Andersen et al., 1994); however, the unaffected serum LPS values in the lambs might be ascribed to the slight changes in the rumen E. coli population during SARA.
The faecal bacteriology also revealed a significant rising trend in E.
coli population during SARA days. In line with our findings, an increased faecal E. coli count was recorded in cows on a SARA-inducing diet (McCann et al., 2016;Plaizier et al., 2017b). The number of faecal E. coli significantly increased in the studied lambs on the third day of SARA (4 ± 2 × 10 7 CFU/g) compared to day 0 (1.40 ± 0.70 × 10 7 ). In agreement with this finding, Diez-Gonzalez et al. (1998) reported an increase in E. coli count from 2 × 10 4 to 6.30 × 10 6 CFU/g of colon contents in cows with SARA. Similarly, a 1000-fold decline was also detected in E.
coli population in hindgut when the cattle were switched from a highcorn diet to a forage diet within 5 days (Callaway et al., 2009). Based on the results, it seems that during SARA, E. coli proliferation occurs more often in the colon than in rumen. This finding is further supported by Emmanuel et al. (2007) who found that LPS was mainly absorbed from the colons in cows on a SARA-inducing diet.
The SARA induction resulted in a significant rising trend (from 0.30 × 10 7 to 3.60 × 10 7 CFU/g) in the number of faecal LAB community in the studied lambs. This suggests that the bacterial community might have a prominent metabolic potential to transfer from forage to high-concentrate diets with the occurrence of SARA. Consistent with our results, it was shown that the Lactobacillales bacterial order was a predominant faecal flora in cows on a SARA-inducing diet (Mao et al., 2012). The SGD including Enterococcus spp. and Streptococcus bovis are mainly lactic acid producers and common intestinal and faecal flora in ruminants that can also involve in the LAB category (Devriese et al., 1992;Ghali et al., 2004). Despite the falling trend in SGD organisms in the rumen, its faecal count showed a nonsignificant rising trend during SARA in the studied lambs. Incompatible with our results, performing a SARA-inducing challenge reduced the number of Streptococcus bovis in caecal digesta and faeces of dairy cows (Plaizier et al. (2017b). The differences in species (sheep versus cow), diagnostic techniques (culture-based versus molecular methods), consumed concentrate (crushed corn versus wheat and barley), and SARA duration might explain this discrepancy. Moreover, it has been reported that compared with rumen pH perturbation, the bacterial population changes during SARA in sheep were more stable and valuable for SARA screening (F. Li et al., 2017). Therefore, understanding the changes in rumen and faecal bacterial populations in lambs on a SARA-inducing diet might be conducive to identifying the risk prediction or diagnosis of SARA, establishing appropriate nutritional strategies, and applying in situ treatment and control methods in sheep flock.

CONCLUSIONS
As an interesting finding, SARA in the lambs did not increase serum LPS value; it may be attributed to the short duration of SARA in the studied lambs or probable high clearance of LPS by the liver in the lambs. However, this necessitates more research for a more definitive conclusion.
The increased rumen (4.10 ± 2.80 × 10 7 CFU/ml) and faecal (3.60 ± 2.50 × 10 7 CFU/g) LAB can be useful indicators for improving the nutritional strategy, developing flock health practice, and screening SARA in lambs. In addition, SARA had no significant effects on blood LPS and lactate levels in the lambs.
Given the strong positive correlation observed between the rumen pH and the faecal pH changes, a reduced faecal pH at 6.30 ± 0.20 level could also be helpful for predicting SARA risk in lambs.

CONFLICTS OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
Data are available upon reasonable request to the corresponding author.